Our long term objective is to develop potent and selective inhibitors of the picornaviral processing enzymes that may be of value in antiviral therapy. To achieve this goal, we will study the catalytic and binding properties of the 3C cysteine proteinases of hepatitis A virus, human rhinovirus types 2 and 14 and human poliovirus. The collaborating group, led by Bruce Malcolm at Protos, has expressed large quantities of the hepatitis enzyme. We have examined a variety of potential peptide sub- strates and developed rapid, quantitative assays. We are now poised to expand our study by refining the peptide structure that is recognized by this enzyme, by developing new assay methods based on sensitive fluorescence methods, and by constructing potential inhibitors based on peptide structures. We will utilize all these methods to thoroughly analyze the interaction of substrates and inhibitors with the Hepatitis enzyme. As the hepatitis enzyme is in hand, it will serves as our initial target. However, we will pursue the closely related rhinovirus and poliovirus enzymes by the same approach. We anticipate that each system will provide new insights into selective inhibitors design. The human cysteine proteinases found in the lysosomal compartment of most human cells, cathepsins B,H, and L, will be utilized in studies for the specificity of compounds designed against the picornaviral proteinases. Samples of enzymes and first-generation inhibitor will be provided to a collaborating crystallography group. Data arising from this approach will be utilized to design newer inhibiotrs in a further effort to improve the selectivity.